Leishmaniasis is an important human infectious disease that afflicts over 15 million people worldwide and causes significant morbidity or death if the visceral form is left untreated. In the mammalian host, Leishmania infect phagocytic cells wherein they reside and replicate in vacuolar compartments called Leishmania parasitophorous vacuoles (LPVs). LPVs undoubtedly play a complex role in the infection as they must provide protection for the parasite; allow nutrient acquisition by the parasite; and be a conduit for parasite strategies that modulate host cell activities. Although our current understanding of the composition and functions of LPVs incomplete, my group has presented recent evidence that LPVs maintain continuous interactions with the endocytic pathway as well as the secretory pathway. We therefore assert that elucidation of the molecular composition of LPVs will not only shed light on processes that contribute to the survival of Leishmania in infected cells but also that they will shed light on the mechanisms that several intracellular pathogens exploit to subvert host cell processes. The long-term goal of our work is to identify pathways that could be targeted for inhibition of the intracellular growth of Leishmania as there are limited therapies to control this important parasite. The goal of the studies proposed here is to document the spatio-temporal composition of LPVs. Specifically, we propose to isolate LPVs that harbor Leishmania donovani parasites at several times post infection of cells grown in the presence or absence of stable isotopes of amino acids. My group has developed an LPV affinity purification strategy that makes this goal feasible. The LPV preparations will be fractionated on the basis of physical properties of the molecules therein to manage their complexity. Molecules in the LPV sub-fractions will be identified using a customized multidimensional protein identification approach which will include fractionation on a strong cation exchange column followed by further fractionation on a long reverse phase column that is coupled to a LTQ Orbitrap XL System for MS/MS peptide identification. This scheme should achieve high proteome coverage. By performing infections in cells that are grown in stable isotopes of amino acids, the abundance of LPV molecules over the course of infection will be tracked. In addition to elucidating the proteome of Leishmania parasites in infected cells, this study will also identify host cell molecules that traffic to LPVs. These latter molecules are likely to play important roles in the transmission of signals aimed at modulating host cell processes. My group is particularly well suited to this task because our previous studies on the biology of Leishmania infections have already produced new information on the composition and characteristics of this dynamic compartment.